In 1975, Kohler and Milstein reported the establishment of a continuous hybrid cell line (hybridoma) derived by the fusion of murine myeloma cells to spleen cells from an immunized mouse which secreted monoclonal antibody to sheep red blood cells; Nature, Volume 256, 495 (1975). Numerous publications have since appeared describing the production of monoclonal antibodies to other antigens and haptens. See, for example, Current Topics in Microbiology and Immunology, Volume 81, F. Melchers, M. Potter, and N. Warner, ed., Springer-Verlag, 1978, and references contained therein; and Monoclonal Antibodies, R. Kennett, T. McKearn and K. Bechtol, ed., Plenum Press, 1980, and references contained therein.
Although the general technique of producing hybridomas is well known and understood, there are still considerable difficulties involved in producing and selecting a hybridoma cell line secreting antibody having a given set of desired properties.
European Patent Application No. 25,722, published Mar. 25, 1981, discloses the production of a monoclonal antibody to a human T-lymphocyte cell surface antigen.
The production of monoclonal antibodies to the cardiac glycoside, digoxin, has been reported in Federation Proceedings, Volume 39, 928 (1980) and in Scand. J. clin. Lab. Invest., Volume 41, 75 (1981).
There is a rapidly expanding market for clinical diagnostic assays which can be used to monitor the levels of various therapeutic drugs in body fluids. The anti-asthmatic agent, theophylline, is a drug whose therapeutic range is very narrow. An immunoassay for theophylline requires a highly specific antibody because of the occurrence in body fluids of other xanthines which are closely related structurally to theophylline and which, if recognized by the anti-theophylline antibody, would produce an erroneous value for the theophylline concentration in the fluid being analyzed. Theophylline is 1,3-dimethylxanthine while four of the most commonly encountered cross-reactive xanthines are: caffeine, 1,3,7-trimethylxanthine; theobromine, 3,7-dimethylxanthine; xanthine; and hypoxanthine. The most frequently encountered xanthine which is a potential cross-reactant is caffeine which is ubiquitous in popular beverages. It is also a metabolite of theophylline in neonates in whom the level of caffeine may approach the level of theophylline. It is thus essential that anti-theophylline antibodies employed in a diagnostic immunoassay for theophylline not cross-react with caffeine.
Theophylline and other compounds of formula weight generally less than 1000 are not immunogenic unless coupled to a carrier which is itself immunogenic; see, for example, H. N. Eisen, Immunology, Harper and Row, 1980. Such compounds are called haptens and there are numerous methods known in the art for coupling them to carriers in order to render the hapten immunogenic.
The choice of carrier and the site of attachment of the hapten to the carrier are known to influence the immunogenicity of the hapten-carrier conjugate as well as the specificity of the antibodies produced. See, for example, B. F. Erlanger, Methods in Enzymology, Volume 70, 85 (1980).
U.S. Pat. No. 4,156,081, issued May 22, 1979, to Singh, et al. describes the synthesis of 3-substituted theophylline derivatives and their use as immunogens to produce antibodies to theophylline which do not cross-react with caffeine. They do, however, cross-react with 1-methylxanthine. Furthermore, there are large quantities of antiserum needed for a commercial immunoassay and their production by animal immunization is slow, laborious and not readily reproducible from animal-to-animal or even bleed-to-bleed in the same animal.
European Patent Application No. 44,441, published Jan. 27, 1982, discloses the production of monoclonal antibodies to drugs. It does not disclose a monoclonal antibody to theophylline which substantially lacks cross-reactivity with caffeine.